In particular the present invention relates to a process for synthesizing steroid-type compounds hydroxylated in a position a to a double bond.
It also relates to the use of molecules thus synthesized as cyclotoxic agents active against cells which have a high proliferative potential.
The steroids are molecules which comprise a cyclopentanophenanthrene skeleton, widely distributed in the living world, and which have great physiological significance. This structure is thus found in bile acids, sex or cortical hormones, certain vitamins, and cardenolides of plant origin.
A great number of them have a therapeutic potential or properties, and it is thus particularly useful to have available a simple and inexpensive process for synthesizing them. A key stage of this synthesis consists of the selective hydroxylation of certain positions of the skeleton.
Among the steroids which are of significance, the cardenolides constitute a group of steroids having 23 carbons which exist in the form of glucosides in various plants, mainly in the foxglove, lily and corn crowfoot families. Many of these glucosides have a considerable cardiotonic activity and some are used in therapeutics, especially digitalin, digitoxin and digoxin. Schematically, the aglucon part of these molecules is characterized by the presence of a xe2x80x9cbutenolidexe2x80x9d-type (unsaturated lactone) side chain, a 14-hydroxyl in the cis position with respect to the 18 methyl and a 3-hydoxyl [sic]. For example, this structure is found in the digitoxigenin molecule of formula: 
The sugars which form a glucoside with the steroid via a bond established with the C-3 hydroxyl comprise glucose and rhamnose, as well a certain number of unusual sugars, such as 2,6-dideoxyhexose, often methylated in the C-3 position.
The way to construct an xcex1,xcex2, lactone [sic] on C17 [lacuna] cyclic structure necessary for the pharmacological activity of this type of compound has been described, especially by Marini-Bettolo et al., (Can. J. Chem. 1981, 59, 1403-1404). Conventionally, 17-keto-xcex1,xcex2-ene derivatives are used as starting compound and lithium ethoxyacetalide [sic], lithium xcex2-furyl and lithium 2-methoxyfuryl are used as reactants. These processes of synthesis cannot be used on the industrial scale because they have a certain number of disadvantages. The time required to obtain the product is relatively long and, moreover, the synthesis is carried out under severe conditions (organic solvents, high temperature). Moreover, the yield of the reaction is low (of the order of 10%) and the purification of the product is difficult, in view of the many additional products which appear during the reaction. Finally, the starting material is both very expensive and very difficult to obtain.
Another group of steroid compounds which has a potential significance in therapeutics consists of the oxysterols, the structure of which derives directly from that of cholesterol via the presence of one or more oxygenated functional groups carried either by the rings or by the side chain of cholesterol. A role in the inhibition of cholesterol synthesis was first attributed to the oxysterols.
The oxysterols were then studied as antiproliferative agents.
In a recent review, Smith and Johnson (Free Radical Biology and Medicine, 1989, 7, 285-332) itemize the mammary cells attacked in vitro by these compounds. In a completely different approach, Cheng et al., (J. Chem. Res., 1977, 217, 2501-2521) have isolated and identified antitumoral active principles from drugs used in the traditional Chinese pharmacopoeia.
Thus the drug Bombix Cum Botryte [sic] contains 7xcex2-hydroxycholesterol which has a cytotoxic activity against cultured cells showing significant multiplication, such as lymphoma (mouse RDM4 and YAC-1) (Cancer Biochem. Biophys., 1986, 9, 75-83), HTC cells (rat liver tumoral cells) (Biochem. Biophys. Res. Commun., 1984, 120, 192-198), rat fibroblasts originating from the heart (C.R. Acad. Sci. Paris, 1984, 299, 221-225) and rat liver epithelial cells (Cell Biol. Toxicol., 1989, 5, 261-270). On the other hand, cells which have a weaker potential for division and which achieve a certain degree of differentiation when cultured, such as cultured heart cells and hepatocytes from newborn rats, are not detrimentally affected by this molecule. The cytotoxicity of 7-hydroxycholesterol [sic] is shown by a halt in cell growth, followed by morphological modifications which precede the detachment and the rapid lysis of the cells.
Rong et al., (C.R. Acad. Sci. Paris, 1985, 300, 89-94) have shown that the sodium salts of 7xcex2-hydroxycholesterol 3,7-bis(hemisuccinate) have an antitumoral activity, injected intraperitoneally into mice carrying ascitic tumors, [lacuna] Krebs-II transplantable carcinoma. It [sic] also shown, in female Sprague-Dawley rats, that (22 R)-cholest-5-ene-3xcex2,7xcex222-triol effectively reduced the development of tumors induced by 7,12-di-methylbenz(xcex1)anthracene [sic] (Iversen et al., Virchows Archiv B, 1986, 51, 313-320).
It is generally assumed that the cytotoxic effect of the oxysterols results from the superimposition of several phenomena, the most important of which are the inhibition of the synthesis of cholesterol by inhibition of a key enzyme (HMGR) and a destabilization of the plasmic membranes. In this context, and with a long-term therapeutic prospective, the inventors have studied the effects and the mechanism of action of 7xcex2-hydroxycholesterol on spontaneously transformed lines obtained from primary cultures of astrocytes from newborn rats (normal cells). In the central nervous system (CNS), the astrocytes (glial cells) have retained the potentiality of multiplying, which phenomenon is manifested in certain pathological states, such as reactional gliosis (inflammation) and formation of glioblastomas (neoplasia).
Now, a recent publication shows that 7xcex2-hydroxycholesterol is metabolized, in transformed astrocytes, to esters of fatty acids.
The conventional route of chemical synthesis used for obtaining 7xcex2-hydroxycholesteryl ester resorts to 7-ketocholesterol as starting material. 
After esterification of the OH in the 3-position, the functional group is reduced with NaBH4. The two a and xcex2, isomers are obtained with a yield of 70% for the xcex2 isomer. This process is difficult to transpose to the industrial level, given its duration.
This is why the present invention relates to a gentle process for the hydroxylation of an acyclic or cyclic methylene radical in the allylic position, characterized in that a peroxidase, iodide ions and hydrogen peroxide (H2O2) are reacted with this methylene radical to produce a compound hydroxylated in a position a to a double bond.
The peroxidase used is preferably lactoperoxidase (LPO), the iodide ions generally arise from KI and the hydrogen peroxide can be generated in situ.
In contrast to the conventional processes for hydroxylation, this process does not involve a powerful reducing agent.
The OH radical is provided by the decomposition of H2O2, catalyzed by lactoperoxidase, an enzyme which is commercially available at reasonable cost.
The reaction can be carried out at temperatures of between 20xc2x0 C. and 40xc2x0 C., and will not affect functional groups present on the molecule.
The present invention thus relates to a process for the hydroxylation of an acyclic or cyclic methylene in the allylic position, characterized in that the starting compound contains at least one unprotected ester functional group, and in that this ester functional group is recovered intact in the compound after hydroxylation.
The present invention also relates to a process for the hydroxylation of an acyclic or cyclic methylene in the allylic position, in which the starting compound contains an ether functional group.
The allylic methylene radical is preferably:
a secondary carbon (1) arranged on [sic] the following Scheme Ia: 
in which the carbon (2) is preferably connected to a hydrogen or to other carbons.
As an example of a compound of this type, there may be mentioned derivatives: 
hydroxylation being carried out on the terminal carbon of the double bond with migration of the latter. This type of compound being able [sic] to contain other substituents, with the proviso that there are no other allylic methylenic carbons.
or else a secondary carbon (1) arranged according to the following Scheme Ib: 
in which (A) is a cyclic structure which is preferably of pregnane type; for example, the methylenic carbon which is in the 7-position of the xcex945-pregnene [sic] cyclic system.
By way of example, there may be mentioned: 
the hydroxylation being carried out at a position xcex1 to the (6-7) double bond. This type of compound being able [sic] to contain other substituents with the proviso that there are no other allylic-methylenic [sic] carbons.
It is thus that the present invention relates to a process for hydroxylation, characterized in that LPO, KI and H2O2 are reacted with xcex9422-23-norcholene [sic] 3-acetate of formula: 
and in that xcex9417-20-norcholene-23-ol [sic] 3-acetate of formula: 
is obtained in a single stage.
xcex9422-23-Norcholene [sic] 3-acetate is obtained from cholanic acid-3-ol. This compound, whose purchasing price is not very high, is easily converted to xcex9422-23-norcholene-3-ol [sic] according to the modified process of Vaida et al., (Tetrahedon Lett., 1968, 50, 5173-5174).
xcex9417-20-Norcholene-23-ol [sic] 3-acetate then makes it possible, by a series of reactions schematicized below, to obtain a cardenolide aglucon. 
This process is simple, requires little energy, its duration is short and its cost price low, in view of the low cost of the reactants. Additionally, the reaction has a good yield and makes it possible to prepare the product on the semi-macro scale, with few additional products.
Indeed, as a result of its specificity, the use of an enzyme as catalyst promotes the production of a major product.
This synthetic route makes it possible to activate the 17,20 and 23 carbons. For this reason, other conversions can be carried out before the final product is obtained. By this process, products having a greater pharmacological activity and lower toxicity can be obtained.
According to another aspect of the invention, the process for hydroxylation is characterized in that a peroxidase, iodide ions and hydrogen peroxide are reacted with a C3 monoester of cholesterol of formula: 
in which R represents a carbon chain corresponding to a fatty acid, and in that an ester of 7-hydroxycholesterol of formula: 
is obtained in the form of a mixture of esters of 7xcex1- and 7xcex2-hydroxycholesterol.
The fatty acids are universal constituents of all the lipid compounds and are in majority containing even numbers of carbon atoms (at least 4) and containing a linear chain. The natural fatty acids can be saturated or unsaturated and generally they then have from 2 to 6 double bonds; there also exist natural fatty acids having triple bonds.
These fatty acids can esterify the alcohol functional group in the 3-position of the cholesterol molecule. In contrast to the monoesters of 7-keto-cholesterol used for the preparation of the hydroxyl derivatives, the monoesters of cholesterol are readily available at a cost which is not very high.
The reaction is performed in a single stage, at a temperature between 20xc2x0 C. and 37xc2x0 C., in a lipophilic organic solvent.
A mixture of xcex1 and xcex2 isomers of 7-hydroxycholesteryl esters is obtained with proportions of 50% and 50% respectively. The yield can be optimized by adaptation of the solvents, concentration of the reactants and the reaction temperature depending on the type of isomer desired.
The present invention also relates to substituted or unsubstituted fatty acid esters at the C3 [sic] position of xcex2-hydroxycholesterol [sic] which can especially be obtained by the process according to the invention. The fatty acids preferably contain from 6 to 24 carbon atoms and can contain one or more unsaturations.
In a preferred aspect of the invention, the starting material is chosen from the group of cholesterol esters comprising cholesterol palmitate, oleate, hexenoate, decenoate and arachidonate.
Palmitic acid is a saturated aliphatic fatty acid of general formula C16 H32O2.
Oleic, hexenoic and decenoic acids are monoethenic fatty acids having respectively, for empirical formula, C18H34O2, C5H10O2 and C10H18O2. Arachidonic acid has four double bonds and corresponds to the formula C20H32O2.
For cytotoxic and antitumoral applications, in the process for the hydroxylation according to the invention of a C3 monoester of cholesterol, the 7xcex2 isomer of the hydroxycholesteryl ester obtained is isolated from the reaction mixture, the said isomer belonging to the group comprising especially the esters of palmitic, oleic, hexenoic, decenoic and arachidonic acid.
According to another of its aspects, the process for hydroxylation consists in reacting a peroxidase, iodide ions and hydrogen peroxide with a C3 ether of 
in which R represents a carbon chain corresponding to a fatty acid, to obtain an ether of 7-hydroxycholesterol of formula: 
Preferably, the 7xcex2 isomer of the hydroxycholesteryl ether obtained is isolated from the reaction mixture, the said isomer belonging in particular to the group comprising the ethers of palmitic, oleic, hexenoic, decenoic and arachidonic acid.
These compounds can also be synthesized from 7-ketocholesterol and the alkenyl chloride corresponding to the fatty acid, for example CH3xe2x80x94(CH2)7xe2x80x94CHxe2x95x90CHxe2x80x94(CH2)7xe2x80x94CH2Cl for oleic acid. A reduction by NaH4 is then carried out.
The formation of an ether between the carbon chain of the fatty acid and the steroid molecule, in place of an ester functional group, gives a more stable molecule which can resist hydrolysis, especially by hepatic enzymes, and can thus be injected peripherally.
This is why the subject of the present invention is a pharmaceutical composition, characterized in that it contains at least one C3-OH ether of 7xcex2-hydroxycholesterol chosen from the group comprising the ethers of palmitic, oleic, hexenoic, decenoic and arachidonic acid, and a vehicle acceptable for its administration.
Such a composition is useful as a cytotoxic agent on cells which have a high proliferative potential.
The present invention also relates to a pharmaceutical composition, characterized in that it contains at least one C3 fatty acid monoester of 7xcex2-hydroxycholesterol preferably chosen from the group comprising the palmitate, oleate, hexenoate, decenoate and arachidonate, and a vehicle acceptable for its administration.
In particular, the present invention relates to the use of a C3 [sic] monoester of 7xcex2-hydroxycholesterol and of a fatty acid, such as defined above, for the preparation of a pharmaceutical composition intended for the treatment of cells having a high proliferative potential. Indeed, this composition is useful as a cytotoxic agent.
According to another aspect of the invention, a C7 ester of the 7xcex2-hydroxylcholesteryl [sic] ester described above is used. In the case where more hydrophilic compounds are desired, a particularly advantageous molecule is represented by cholesteryl 7-phosphoenolpyruvate 3-oleate.
The liposoluble compounds of 7xcex2-hydroxycholesterol can be put into the form of liposomes or of a combination with phospholipids, such as those used in the preparation of the liposomes, and especially phosphatidylcholine. They can also be used in combination, in the liposomes, with GM1- and GT1b-type gangliosides. This combination between gangliosides and C3 esters of 7xcex2-hydroxycholesterol makes it possible to obtain a vector which is smaller in size and more hydrophilic.
These liposomes will be administered parenterally with an acceptable vehicle.
These compositions have a great affinity for nervous tissue which is rich in lipids.
The compositions containing an ether or an ester of xcex2-hydroxycholesterol and of fatty acid, incorporated in liposomes containing phosphatidylcholine and, for example, monosialoganglioside (GM1), are thus particularly useful as antitumoral [lacuna] on intracerebral glioblastoma-type tumors of the nervous system, or as an agent for the treatment of reactional glioses.
The present invention thus relates to the preparation of a pharmaceutical composition containing at least one C3 monoester of 7xcex2-hydroxycholesterol in the form of liposomes, and useful as an antitumoral agent. According to one aspect of the invention, this pharmaceutical composition is characterized in that it is useful on tumors of the nervous system, in particular glioblastomas. The injection of liposomes containing phosphatidylcholine and certain esters of 7xcex2-hydroxycholesterol causes significant regression of the subcutaneous glioblastomas induced in rats.
According to another aspect of the invention, the pharmaceutical composition containing a monoester of 7xcex2-hydroxycholesterol in the form of liposomes is used as an anti-inflammatory agent of the nervous system, in particular in reactional glioses.
The examples which follow are intended to illustrate the invention without in any way limiting its scope.